Mouse Hepatitis Virus Type Research Articles

Type I IFNs: A Blessing in Disguise or Partner in Crime in MERS-CoV-, SARS-CoV-, and SARS-CoV-2-Induced Pathology and Potential Use of Type I IFNs in Synergism with IFN-γ as a Novel Antiviral Approach Against COVID-19.

Since the end of 2019, the emergence of novel coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has accelerated the research on host immune responses toward the coronaviruses. When there is no approved drug or vaccine to use against these culprits, host immunity is the major strategy to fight such infections. Type I interferons are an integral part of the host innate immune system and define one of the first lines of innate immune defense against viral infections. The in vitro antiviral role of type I IFNs against Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV (severe acute respiratory syndrome coronavirus) is well established. Moreover, the involvement of type I IFNs in disease pathology has also been reported. In this study, we have reviewed the protective and the immunopathogenic role of type I IFNs in the pathogenesis of MERS-CoV, SARS-CoV, and SARS-CoV-2. This review will also enlighten the potential implications of type I IFNs for the treatment of COVID-19 when used in combination with IFN-γ.

Toll-like receptor-2 exacerbates murine acute viral hepatitis.

SummaryViral replication in the liver is generally detected by cellular endosomal Toll‐like receptors (TLRs) and cytosolic helicase sensors that trigger antiviral inflammatory responses. Recent evidence suggests that surface TLR2 may also contribute to viral detection through recognition of viral coat proteins but its role in the outcome of acute viral infection remains elusive. In this study, we examined in vivo the role of TLR2 in acute infections induced by the highly hepatotrophic mouse hepatitis virus (MHV) type 3 and weakly hepatotrophic MHV‐A59 serotype. To address this, C57BL/6 (wild‐type; WT) and TLR2 knockout (KO) groups of mice were intraperitoneally infected with MHV3 or MHV‐A59. MHV3 infection provoked a fulminant hepatitis in WT mice, characterized by early mortality and high alanine and aspartate transaminase levels, histopathological lesions and viral replication whereas infection of TLR2 KO mice was markedly less severe. MHV‐A59 provoked a comparable mild and subclinical hepatitis in WT and TLR2 KO mice. MHV3‐induced fulminant hepatitis in WT mice correlated with higher hepatic expression of interferon‐β, interleukin‐6, tumour necrosis factor‐α, CXCL1, CCL2, CXCL10 and alarmin (interleukin‐33) than in MHV‐A59‐infected WT mice and in MHV3‐infected TLR2 KO mice. Intrahepatic recruited neutrophils, natural killer cells, natural killer T cells or macrophages rapidly decreased in MHV3‐infected WT mice whereas they were sustained in MHV‐A59‐infected WT mice and MHV3‐infected TLR2 KO. MHV3 in vitro infection of macrophagic cells induced rapid and higher viral replication and/or interleukin‐6 induction in comparison to MHV‐A59, and depended on viral activation of TLR2 and p38 mitogen‐activated protein kinase. Taken together, these results support a new aggravating inflammatory role for TLR2 in MHV3‐induced acute fulminant hepatitis.

Mouse Hepatitis Virus Infection Induces a Toll-Like Receptor 2-Dependent Activation of Inflammatory Functions in Liver Sinusoidal Endothelial Cells during Acute Hepatitis.

Under physiological conditions, the liver sinusoidal endothelial cells (LSECs) mediate hepatic immune tolerance toward self or foreign antigens through constitutive expression of anti-inflammatory mediators. However, upon viral infection or Toll-like receptor 2 (TLR2) activation, LSECs can achieve proinflammatory functions, but their role in hepatic inflammation during acute viral hepatitis is unknown. Using the highly virulent mouse hepatitis virus type 3 (MHV3) and the attenuated variants 51.6-MHV3 and YAC-MHV3, exhibiting lower tropism for LSECs, we investigated in vivo and in vitro the consequence of LSEC infection on their proinflammatory profiles and the aggravation of acute hepatitis process. In vivo infection with virulent MHV3, in comparison to attenuated strains, resulted in fulminant hepatitis associated with higher hepatic viral load, tissue necrosis, and levels of inflammatory mediators and earlier recruitment of inflammatory cells. Such hepatic inflammatory disorders correlated with disturbed production of interleukin-10 (IL-10) and vascular factors by LSECs. We next showed in vitro that infection of LSECs by the virulent MHV3 strain altered their production of anti-inflammatory cytokines and promoted higher release of proinflammatory and procoagulant factors and earlier cell damage than infection by attenuated strains. This higher replication and proinflammatory activation in LSECs by the virulent MHV3 strain was associated with a specific activation of TLR2 signaling by the virus. We provide evidence that TLR2 activation of LSCEs by MHV3 is an aggravating factor of hepatic inflammation and correlates with the severity of hepatitis. Taken together, these results indicate that preservation of the immunotolerant properties of LSECs during acute viral hepatitis is imperative in order to limit hepatic inflammation and damage. Viral hepatitis B and C infections are serious health problems affecting over 350 million and 170 million people worldwide, respectively. It has been suggested that a balance between protection and liver damage mediated by the host's immune response during the acute phase of infection would be determinant in hepatitis outcome. Thus, it appears crucial to identify the factors that predispose in exacerbating liver inflammation to limit hepatocyte injury. Liver sinusoidal endothelial cells (LSECs) can express both anti- and proinflammatory functions, but their role in acute viral hepatitis has never been investigated. Using mouse hepatitis virus (MHV) infections as animal models of viral hepatitis, we report for the first time that in vitro and in vivo infection of LSECs by the pathogenic MHV3 serotype leads to a reversion of their intrinsic anti-inflammatory phenotype toward a proinflammatory profile as well to as disorders in vascular factors, correlating with the severity of hepatitis. These results highlight a new virus-promoted mechanism of exacerbation of liver inflammatory response during acute hepatitis.

Evaluation of antiviral activities of Houttuynia cordata Thunb. extract, quercetin, quercetrin and cinanserin on murine coronavirus and dengue virus infection.

ObjectiveTo evaluate the in vitro activities of the ethyl acetate (EA) fraction of Houttuynia cordata (H. cordata) Thunb. (Saururaceae) and three of its constituent flavonoids (quercetin, quercitrin and rutin) against murine coronavirus and dengue virus (DENV).MethodsThe antiviral activities of various concentrations of the EA fraction of H. cordata and flavonoids were assessed using virus neutralization tests against mouse hepatitis virus (MHV) and DENV type 2 (DENV-2). Cinanserin hydrochloride was also tested against MHV. The EA fraction of H. cordata was tested for acute oral toxicity in C57BL/6 mice.ResultsThe EA fraction of H. cordata inhibited viral infectivity up to 6 d. Cinanserin hydrochloride was able to inhibit MHV for only 2 d. The 50% inhibitory concentrations (IC50) of the EA fraction of H. cordata added before the viral adsorption stage were 0.98 μg/mL for MHV and 7.50 μg/mL for DENV-2 with absence of cytotoxicity. The mice fed with the EA fraction up to 2 000 mg/kg did not induce any signs of acute toxicity, with normal histological features of major organs. Certain flavonoids exhibited comparatively weaker antiviral activity, notably quercetin which could inhibit both MHV and DENV-2. This was followed by quercitrin which could inhibit DENV-2 but not MHV, whereas rutin did not exert any inhibitory effect on either virus. When quercetin was combined with quercitrin, enhancement of anti-DENV-2 activity and reduced cytotoxicity were observed. However, the synergistic efficacy of the flavonoid combination was still less than that of the EA fraction.ConclusionsThe compounds in H. cordata contribute to the superior antiviral efficacy of the EA fraction which lacked cytotoxicity in vitro and acute toxicity in vivo. H. cordata has much potential for the development of antiviral agents against coronavirus and dengue infections.

Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells.

Coronaviruses (CoVs) have shown neuroinvasive properties in humans and animals secondary to replication in peripheral organs, but the mechanism of neuroinvasion is unknown. The major aim of our work was to evaluate the ability of CoVs to enter the central nervous system (CNS) through the blood-brain barrier (BBB). Using the highly hepatotropic mouse hepatitis virus type 3 (MHV3), its attenuated variant, 51.6-MHV3, which shows low tropism for endothelial cells, and the weakly hepatotropic MHV-A59 strain from the murine coronavirus group, we investigated the virus-induced dysfunctions of BBB in vivo and in brain microvascular endothelial cells (BMECs) in vitro. We report here a MHV strain-specific ability to cross the BBB during acute infection according to their virulence for liver. Brain invasion was observed only in MHV3-infected mice and correlated with enhanced BBB permeability associated with decreased expression of zona occludens protein 1 (ZO-1), VE-cadherin, and occludin, but not claudin-5, in the brain or in cultured BMECs. BBB breakdown in MHV3 infection was not related to production of barrier-dysregulating inflammatory cytokines or chemokines by infected BMECs but rather to a downregulation of barrier protective beta interferon (IFN-β) production. Our findings highlight the importance of IFN-β production by infected BMECs in preserving BBB function and preventing access of blood-borne infectious viruses to the brain. Coronaviruses (CoVs) infect several mammals, including humans, and are associated with respiratory, gastrointestinal, and/or neurological diseases. There is some evidence that suggest that human respiratory CoVs may show neuroinvasive properties. Indeed, the severe acute respiratory syndrome coronavirus (SARS-CoV), causing severe acute respiratory syndrome, and the CoVs OC43 and 229E were found in the brains of SARS patients and multiple sclerosis patients, respectively. These findings suggest that hematogenously spread CoVs may gain access to the CNS at the BBB level. Herein we report for the first time that CoVs exhibit the ability to cross the BBB according to strain virulence. BBB invasion by CoVs correlates with virus-induced disruption of tight junctions on BMECs, leading to BBB dysfunction and enhanced permeability. We provide evidence that production of IFN-β by BMECs during CoV infection may prevent BBB breakdown and brain viral invasion.

Depletion of Alveolar Macrophages Ameliorates Virus-Induced Disease following a Pulmonary Coronavirus Infection

Coronaviruses cause respiratory disease in humans that can range from mild to severe. However, the pathogenesis of pulmonary coronavirus infections is poorly understood. Mouse hepatitis virus type 1 (MHV-1) is a group 2 coronavirus capable of causing severe morbidity and mortality in highly susceptible C3H/HeJ mice. We have previously shown that both CD4 and CD8 T cells play a critical role in mediating MHV-1-induced disease. Here we evaluated the role of alveolar macrophages (AM) in modulating the adaptive immune response and subsequent disease. Depletion of AM using clodronate liposomes administered prior to MHV-1 infection was associated with a significant amelioration of MHV-1-induced morbidity and mortality. AM depletion resulted in a decreased number of virus-specific CD4 T cells in the lung airways. In addition, a significant increase in the frequency and total number of Tregs in the lung tissue and lung airways was observed following MHV-1 infection in mice depleted of AM. Our results indicate that AM play a critical role in modulating MHV-1-induced morbidity and mortality.

Noninvasive molecular imaging of interferon beta activation in mouse liver

Interferon beta (IFN-β) is the priming cytokine in the interferons (IFNs) response that plays essential roles in innate immune system. Only very few studies on IFN activation in animals have been reported before, therefore, we embarked to develop a novel method to dynamically examine IFN-β activation in mouse liver by noninvasive molecular imaging. Interferon beta promoter-directed firefly luciferase gene was integrated into chromosomes of hepatocytes by hydrodynamic injection. Mouse hepatitis virus type 3 (MHV-3) and polyinosinic-polycytidylic acid [poly(I:C)] were used to stimulate the activation of IFN-β. Luciferase activity was used as an indicator of the IFN-β promoter activity in vitro and in vivo. The expression level of IFN-β in the sera and firefly luciferase in the liver was assessed by ELISA and bioluminescence imaging respectively. Western blot was used for detecting proteins expression. A rapid and elevated luciferase expression in the mouse liver induced by poly (I:C) and MHV-3 was detected by bioluminescence imaging. The detectable level of IFN-β in the sera was not induced by MHV-3. Moreover, IFN-β activation was significantly inhibited by the hepatitis C virus (HCV) NS3/4A protease in mouse liver. These results were consistent with IFN-β production in the sera. Therefore, a novel visual method to monitor IFN-β promoter activity was established in the current study. This novel sensitive method can be used for not only assessing IFN-β activation or inhibition in the liver under different conditions, but also screening drug candidates of stimulating or inhibiting of IFN-β production.

プロテアーゼ依存的なコロナウイルス細胞侵入

Previous studies have demonstrated that the SARS-CoV S protein requires proteolytic cleavage by elastase, cathepsin or TMPRSS2 for S-mediated cell-cell or virus-cell membrane fusion. Activation of viral glycoprotein (GP) by protease also has been reported for influenza virus. The most distinctive difference between influenza virus and SARS-CoV is the stage during virus replication in which viral glycoproteins are cleaved by proteases. In influenza virus, the protease makes a simple cut in the GP during maturation. In contrast, SARS-CoV S protein is cleaved by the protease following receptor-induced conformational changes. The protease cleavage site in S protein is thought to be exposed only after receptor binding. In support of this model, we reported that the S protein of mouse hepatitis virus type 2 (MHV-2), which is highly similar to the S protein of SARS-CoV, requires two-step conformational changes mediated by sequential receptor binding and proteolysis to be activated for membrane fusion. Such a mechanism allows for tight temporal control over fusion by protecting the activating cleavage site from premature proteolysis yet allowing efficient cleavage upon binding to the receptor on target cells.

Protective and Pathologic Roles of the Immune Response to Mouse Hepatitis Virus Type 1: Implications for Severe Acute Respiratory Syndrome

Intranasal mouse hepatitis virus type 1 (MHV-1) infection of mice induces lung pathology similar to that observed in severe acute respiratory syndrome (SARS) patients. However, the severity of MHV-1-induced pulmonary disease varies among mouse strains, and it has been suggested that differences in the host immune response might account for this variation. It has also been suggested that immunopathology may represent an important clinical feature of SARS. Little is known about the host immune response to MHV-1 and how it might contribute to some of the pathological changes detected in infected mice. In this study we show that an intact type I interferon system and the adaptive immune responses are required for controlling MHV-1 replication and preventing morbidity and mortality in resistant C57BL/6J mice after infection. The NK cell response also helps minimize the severity of illness following MHV-1 infection of C57BL/6J mice. In A/J and C3H/HeJ mice, which are highly susceptible to MHV-1-induced disease, we demonstrate that both CD4 and CD8 T cells contribute to morbidity during primary infection, and memory responses can enhance morbidity and mortality during subsequent reexposure to MHV-1. However, morbidity in A/J and C3H/HeJ mice can be minimized by treating them with immune serum prior to MHV-1 infection. Overall, our findings highlight the role of the host immune response in contributing to the pathogenesis of coronavirus-induced respiratory disease.

Two-Step Conformational Changes in a Coronavirus Envelope Glycoprotein Mediated by Receptor Binding and Proteolysis

The coronaviruses mouse hepatitis virus type 2 (MHV-2) and severe acute respiratory syndrome coronavirus (SARS-CoV) utilize proteases to enter host cells. Upon receptor binding, the spike (S) proteins of both viruses are activated for membrane fusion by proteases, such as trypsin, present in the environment, facilitating virus entry from the cell surface. In contrast, in the absence of extracellular proteases, these viruses can enter cells via an endosomal pathway and utilize endosomal cathepsins for S protein activation. We demonstrate that the MHV-2 S protein uses multistep conformational changes for membrane fusion. After interaction with a soluble form of the MHV receptor (CEACAM1a), the metastable form of S protein is converted to a stable trimer, as revealed by mildly denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liposome-binding assays indicate that the receptor-bound virions are associated with the target membrane through hydrophobic interactions. The exposure of receptor-bound S protein to trypsin or cathepsin L (CPL) induces the formation of six-helix bundles (6HB), the final conformation. This trypsin- or CPL-mediated conversion to 6HB can be blocked by a heptad repeat peptide known to block the formation of 6HB. Although trypsin treatment enabled receptor-bound MHV-2 to enter from the cell surface, CPL failed to do so. Interestingly, consecutive treatment with CPL and then chlorpromazine enabled a portion of the virus to enter from cell surface. These results suggest that trypsin suffices for the induction of membrane fusion of receptor-primed S protein, but an additional unidentified cellular factor is required to trigger membrane fusion by CPL.

A synergistic interferon‐γ production is induced by mouse hepatitis virus in interleukin‐12 (IL‐12)/IL‐18‐activated natural killer cells and modulated by carcinoembryonic antigen‐related cell adhesion molecules (CEACAM) 1a receptor

The production of interferon-γ (IFN-γ) by infiltrating natural killer (NK) cells in liver is involved in the control of mouse hepatitis virus (MHV) infection. The objectives of this study were to identify the mechanisms used by MHV type 3 to modulate the production of IFN-γ by NK cells during the acute hepatitis in susceptible C57BL/6 mice. Ex vivo and in vitro experiments revealed that NK cells, expressing carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 1a (the MHV receptor), can produce a higher level of IFN-γ in the presence of both L2-MHV3 and interleukin-12 (IL-12)/IL-18. The synergistic production of IFN-γ by NK cells depends on viral replication rather than viral fixation only, because it is inhibited or not induced in cells infected with ultraviolet-inactivated viruses and in cells from Ceacam1a−/− mice infected with virulent viruses. The synergistic IFN-γ production involves the p38 mitogen-activated protein kinase (MAPK) rather than the extracellular signal-regulated kinase-1/2 MAPK signalling pathway. However, the signal triggered through the engagement of CEACAM1a decreases the production of IFN-γ, when these molecules are cross-linked using specific monoclonal antibodies. These results suggest that control of acute hepatitis by IFN-γ-producing NK cells may depend on both production of IL-12 and IL-18 in the liver environment and viral infection of NK cells.

Mouse Hepatitis Virus Type 2 Enters Cells through a Clathrin-Mediated Endocytic Pathway Independent of Eps15

It has recently been shown that cell entry of mouse hepatitis virus type 2 (MHV-2) is mediated through endocytosis (Z. Qiu et al., J. Virol. 80:5768-5776, 2006). However, the molecular mechanism underlying MHV-2 entry is not known. Here we employed multiple chemical and molecular approaches to determine the molecular pathways for MHV-2 entry. Our results showed that MHV-2 gene expression and infectivity were significantly inhibited when cells were treated with chemical and physiologic blockers of the clathrin-mediated pathway, such as chlorpromazine and hypertonic sucrose medium. Furthermore, viral gene expression was significantly inhibited when cells were transfected with a small interfering RNA specific to the clathrin heavy chain. However, these treatments did not affect the infectivity and gene expression of MHV-A59, demonstrating the specificity of the inhibitions. In addition, overexpression of a dominant-negative mutant of caveolin 1 did not have any effect on MHV-2 infection, while it significantly blocked the caveolin-dependent uptake of cholera toxin subunit B. These results demonstrate that MHV-2 utilizes the clathrin- but not caveolin-mediated endocytic pathway for entry. Interestingly, when the cells transiently overexpressed a dominant-negative form (DIII) of Eps15, which is thought to be an essential component of the clathrin pathway, viral gene expression and infectivity were unaffected, although DIII expression blocked transferrin uptake and vesicular stomatitis virus infection, which are dependent on clathrin-mediated endocytosis. Thus, MHV-2 entry is mediated through clathrin-dependent but Eps15-independent endocytosis.

Intrahepatic endothelial and Kupffer cells involved in immunosuppressive cytokines and natural killer (NK)/NK T cell disorders in viral acute hepatitis

During acute viral hepatitis, the intrahepatic tolerance sustained by immunosuppressive cytokines such as interleukin (IL)-4, IL-10, transforming growth factor (TGF)-beta and prostaglandin E2 (PGE2), produced by Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC), natural killer (NK) T cells and natural regulatory T cells may be disturbed. NK cells are recruited normally in the liver and produce interferon (IFN)-gamma to control viral replication. The use of mouse hepatitis virus type 3 (MHV3) attenuated variants showing selected tropisms for KC or LSEC have allowed determining their roles in the disturbances of immune tolerance during viral hepatitis. Groups of C57BL/6 mice were infected with the pathogenic L2-MHV3 (KC+, LSEC+), low attenuated 51.6-MHV3 (KC+, LSEC-) or high attenuated CL12-MHV3 (KC-, LSEC-) variants for the first 3 days. Results showed that IL-10, TGF-beta and PGE2 production in the liver decreased in L2-MHV3-infected mice and increased in 51.6-MHV3- and CL12-MHV3-infected mice. The ratio of IFN-gamma/IL-4 in liver decreased in L2-MHV3-infected mice, while it was not (or low) altered in mice infected with the attenuated MHV3 variant mice. Phenotypic analysis of intrahepatic mononuclear cells revealed that apoptotic NK and NK T cells increased in mice infected with the L2-MHV3, but were minor in 51.6-MHV3- and CL12-MHV3-infected mice. The numbers of CD4+ forkhead box P3+ cells increased in the livers from low pathogenic CL12-MHV3 and YAC-MHV3-infected mice. These results indicate that viral permissivity of KC and LSEC is involved in the decrease of IL-10 and PGE2, while KC may play an additional role in the apoptosis of NK and NK T cells during acute viral hepatitis.

Endosomal Proteolysis by Cathepsins Is Necessary for Murine Coronavirus Mouse Hepatitis Virus Type 2 Spike-Mediated Entry

Most strains of murine coronavirus mouse hepatitis virus (MHV) express a cleavable spike glycoprotein that mediates viral entry and pH-independent cell-cell fusion. The MHV type 2 (MHV-2) strain of murine coronavirus differs from other strains in that it expresses an uncleaved spike and cannot induce cell-cell fusion at neutral pH values. We show here that while infection of the prototype MHV-A59 strain is not sensitive to pretreatment with lysosomotropic agents, MHV-2 replication is significantly inhibited by these agents. By use of an A59/MHV-2 chimeric virus, the susceptibility to lysosomotropic agents is mapped to the MHV-2 spike, suggesting a requirement of acidification of endosomes for MHV-2 spike-mediated entry. However, acidification is likely not a direct trigger for MHV-2 spike-mediated membrane fusion, as low-pH treatment is unable to overcome ammonium chloride inhibition, and it also cannot induce cell-cell fusion between MHV-2-infected cells. In contrast, trypsin treatment can both overcome ammonium chloride inhibition and promote cell-cell fusion. Inhibitors of the endosomal cysteine proteases cathepsin B and cathepsin L greatly reduce MHV-2 spike-mediated entry, while they have little effect on A59 entry, suggesting that there is a proteolytic step in MHV-2 entry. Finally, a recombinant virus expressing a cleaved MHV-2 spike has the ability to induce cell-cell fusion at neutral pH values and does not require low pH and endosomal cathepsins during infection. These studies demonstrate that endosomal proteolysis by cathepsins is necessary for MHV-2 spike-mediated entry; this is similar to the entry pathway recently described for severe acute respiratory syndrome coronavirus and indicates that coronaviruses may use multiple pathways for entry.

Antiviral Activity of Limitin against Encephalomyocarditis Virus, Herpes Simplex Virus, and Mouse Hepatitis Virus: Diverse Requirements by Limitin and Alpha Interferon for Interferon Regulatory Factor 1

Limitin has sequence homology with alpha interferon (IFN-alpha) and IFN-beta and utilizes the IFN-alpha/beta receptor. However, it has no influence on the proliferation of normal myeloid and erythroid progenitors. In this study, we show that limitin has antiviral activity in vitro as well as in vivo. Limitin inhibited not only cytopathic effects in encephalomyocarditis virus- or herpes simplex virus (HSV) type 1-infected L929 cells, but also plaque formation in mouse hepatitis virus (MHV) type 2-infected DBT cells. In addition, administration of limitin to mice suppressed MHV-induced hepatitis and HSV-induced death. The antiviral activity may be mediated in part by 2',5'-oligoadenylate synthetase, RNA-dependent protein kinase, and Mx protein, which inhibit viral replication or degrade viral components, because limitin induced their mRNA expression and enzyme activity. While limitin has antiviral activity as strong as that of IFN-alpha in vitro (the concentration that provided 50% inhibition of cytopathic effect is approximately 30 pg/ml), IFN regulatory factor 1 (IRF-1) dependencies for induction of an antiviral state were different for limitin and IFN-alpha. In IRF-1-deficient fibroblasts, a higher concentration of limitin than of IFN-alpha was required for the induction of antiviral activity and the transcription of proteins from IFN-stimulated response element. The unique signals and the fewer properties of myelosuppression suggest that a human homolog of limitin may be used as a new antiviral drug.

STAT1 and STAT3 alpha/beta splice form activation predicts host responses in mouse hepatitis virus type 3 infection.

Signal transducer and activator of transcription 1alpha (STAT1 alpha) is reported to be essential for IFN-gamma and IFN-alpha regulated gene expression, while STAT1 beta, an alternate splice-form, mediates only IFN-alpha-dependent gene expression. STAT3 alpha and STAT3 beta splice forms are also differentially activated in response to cytokines including IL-6 and IL-10. The aim of this study was to investigate whether the STAT activation will predict the host immune response to viral infection and possibly a therapeutic target for the treatment of viral infection. Mouse hepatitis virus type 3 (MHV-3) resistant strain (A/J) and sensitive mouse strains (BalB/cJ) were infected intraperitoneally (i.p.) with 100 plaque form units (pfu) of MHV-3. The mice were sacrificed at the indicated times, and livers and spleens were immediately frozen in liquid nitrogen. Nuclear extracts proteins were detected by immunoblotting. STAT1 and STAT3 activation in spleen increased 24 to 72 hr following MHV-3 infections in both sensitive and resistant mouse strains. However, over this time period, the ratio of activated alpha to beta splice-form for STAT1 and STAT3 increased above 1.0 in resistant A/J mice, while the ratio fell to <0.3 in MHV-3 sensitive Balb/cJ and C3H/HeJ strains. Activated STAT1 alpha/beta and STAT3 alpha/beta ratio in liver were similar in resistant and sensitive mouse strains. Treatment of sensitive Balb/cJ mice with neutralizing anti-TGF-beta antibody could increase the STAT1 alpha/beta ratio to <1.0 in spleens, predicting enhanced rates of survival. These results suggested that ratio of activated STAT1 alpha/beta and STAT3 alpha/beta in mixed leukocytes from spleen predict the outcome to MHV-3 infection, and may be an important marker and therapeutic target for modification of host immune response to virus infection.

Synergistic antiviral effect of a combination of mouse interferon-alpha and interferon-gamma on mouse hepatitis virus.

Although interferon (IFN)‐α and IFN‐γ have been reported to exhibit a synergistic antiviral effect through the different signaling pathways in vitro, their therapeutic efficacy is not well defined in vivo. The current study was carried out to investigate the combined antiviral effect in a model of mouse hepatitis virus Type 2 (MHV‐2) infection, in which fulminant hepatitis is developed. MHV‐2 was injected intraperitoneally into 4‐week‐old ICR mice, IFN or the vehicle was administered intramuscularly for 5 days, and the antiviral effect was evaluated based on survival periods, liver histology, serum alanine transaminase (ALT) levels, and MHV‐2 virus titers in the liver tissues. The animals in the group treated with a combination of IFN‐α and IFN‐γ survived for longer periods than the groups treated with IFN‐α alone and IFN‐γ alone (IFN‐α 103 (IU/mouse)/‐γ 103 vs. IFN‐α 103, P < 0.005; IFN‐α 103/‐γ 103 vs. IFN‐γ 103, P < 0.001). This is consistent with the lower levels of hepatocellular necrosis and serum ALT and the decreased titers of MHV‐2 virus in the liver tissues (48 hr, P < 0.001; 72 hr, P < 0.001). These findings indicate that a combination of IFN‐α and IFN‐γ exhibits a synergistic antiviral effect on MHV‐2 infection. The biology of MHV‐2 is quite different from that of human hepatitis viruses; however, these results suggest the beneficial combined therapy of IFN‐α and IFN‐γ for the treatment of human viral hepatitis. J. Med. Virol. 69:188–194, 2003. © 2003 Wiley‐Liss, Inc.

Liver-specific alpha 2 interferon gene expression results in protection from induced hepatitis.

The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-alpha). However, systemic delivery of r-hIFN-alpha is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-alpha antiviral efficacy, we have explored the therapeutic potential of murine IFN-alpha2 (mIFNalpha2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-alpha2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-alpha2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-alpha2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-alpha2.

Immunodepression induced by Trypanosoma cruzi and mouse hepatitis virus type 3 is associated with thymus apoptosis.

Trypanosoma cruzi-infected mice show disturbance in the peripheral immune system such as polyclonal lymphocyte activation, autoantibody production, and immunosuppression of T lymphocytes. Previous observations in our laboratory showed that some stocks of T. cruzi can be contaminated with mouse hepatitis virus type 3 (MHV-3). Literature has shown that MHV-3 infection induces immunologic disorders characterized by thymic involution with marked cell depletion. However, the effects of interactions between MHV-3 and the parasite on the immune system are not well understood. In the present study specific-pathogen-free CBA mice were inoculated with MHV-3, alone or associated with different stocks of T. cruzi. Concurrent murine virus infection resulted in increased pathogenicity of T. cruzi infection shown by profound thymic atrophy; loss of cortical thymocytes; depletion of Thy1.2+, CD4+, and CD8+ cells; enhancement of in situ labeling of nuclear DNA fragmentation; and eventually, death of the animals. Such lines of evidence show that the mechanism underlying this thymic atrophy is associated with apoptosis. These results also suggest that MHV-3 can account for the increased immunosuppression observed during experimental infection with the parasite.

Intrahepatic alpha beta-TcRintermediate LFA-1high T cells are stimulated during mouse hepatitis viral infection.

Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent animal model for the study of thymic and extrathymic T cell subpopulation disorders induced during the viral hepatitis. To understand local hepatic immune responses, the phenotypes of resident hepatic lymphocytes were determined and compared that of splenic and thymic T cell subpopulations during the acute viral hepatitis induced by MHV3 in susceptible C57BL/6 mice. Single positive (SP) CD4+ or CD8+ cells strongly increased in the liver. A specific cell population, the double positive (CD4+ C8+) cells, normally present in liver and thymic cell preparations, decreased in C57BL/6 mice following the viral infection. alpha beta-TcRintermediate T cells shifted toward alpha beta-TcRhigh T cells in the liver and thymus of infected mice, but not in their spleen. The specific alpha beta-TcRint or high lymphocytes occurring in the liver of MHV3-infected mice expressed higher levels of leukocyte function antigen-1 (LFA-1) and Pgp-1 (CD44) activation markers, suggesting that they were either activated or antigen-experienced during the viral infection. No significant changes in T cell subpopulations were detected in the spleen. These observations suggest that MHV3 infection could induce an early in situ stimulation of resident hepatic T cells, despite a peripheral immunodeficiency in the thymus and spleen.